Lineage-Specific Adaptive Evolution of the Mosquito Fibrinogen-Related Protein FBN30 at a Predicted Parasite-Facing Interface

Fibrinogen-related proteins (FREPs) contribute to mosquito-parasite interactions, yet the evolutionary processes shaping their functional diversification remain poorly resolved. The mosquito protein FBN30 has been implicated in restricting Plasmodium development, but its molecular basis of action is unknown. Here, the study examines the evolutionary history of FBN30 across Anopheles mosquitoes to test whether lineage-specific adaptive evolution has modified its functional properties. Codon-based analyses of FBN30 orthologs from 29 Anopheles species reveal a single episode of strong episodic diversifying selection confined to the Anopheles darlingi lineage. Site-level tests identify a positively selected residue within the conserved fibrinogen-like (FBG) domain. Ancestral sequence reconstruction shows that this site underwent a serine-to-asparagine substitution along the A. darlingi lineage, with structural modeling indicating only modest local effects on protein stability. Using protein-protein docking and binding affinity prediction as a proxy for functional engagement, the study finds that the reconstructed ancestral FBN30 exhibits significantly stronger predicted affinity for Plasmodium falciparum α-tubulin-1 than the extant A. darlingi protein, whereas the derived substitution alone does not account for this difference. These results indicate that evolutionary divergence in FBN30 is associated with reduced predicted engagement at a parasite-facing interface and support a model in which inhibitory mosquito proteins undergo fine-scale adaptive refinement under parasite-mediated selective pressures.